TNF-Related Apoptosis Inducing Ligand (TRAIL) kills tumor cells with little effect on normal tissues and recombinant TRAIL and antibodies that recognize TRAIL receptors are in clinical trials at the University of Colorado and elsewhere. In addition, TRAIL receptor signaling determines the efficiency with which other agents kill tumor cells. However, tumor cells are often resistant to TRAIL and the mechanisms by which this occurs and what this resistance means for tumor progression and clinical outcomes is poorly understood. We recently discovered that one mechanism by which breast and ovarian tumor cells can become selectively resistant to TRAIL is through the increased expression of the homeobox transcription factor Six1. We found that increased Six1 is common, occurring in >60% of metastatic ovarian cancers and 90% of metastatic breast cancers, and associated with poor clinical outcomes. We further found that Six1 expression is sufficient to make non-metastatic tumor cells metastasize in vivo. Because TRAIL signaling is known to suppress metastasis, we hypothesize that Six1 inhibits TRAIL by a specific mechanism and this leads to increased metastasis resulting in poor clinical outcomes in patients and resistance of patient's tumors to TRAIL. To test this hypothesis we propose an integrated project that will determine the molecular mechanism by which Six1 inhibits TRAIL receptor-induced apoptosis, test if these mechanisms are responsible for increased metastasis in mice and determine whether these effects apply in primary tumor cells from patients and if they lead to worse clinical outcomes. Because this work encompasses research on basic mechanisms at the cellular level, testing those mechanisms in animal models of cancer progression and metastasis and clinical and translational studies in ovarian and breast cancer patients'tumors, we have adopted a team approach that will involve a cell biologist with expertise in TRAIL signaling (Dr. Thorburn), a pioneer in the analysis of Six1 in cancer development and progression (Dr. Ford) and an oncologist with expertise in clinical and translational research (Dr. Behbakht). To achieve these goals we have the following aims: 1. Determine how Six1 alters signaling by TRAIL receptor-targeted therapeutic agonists in ovarian and breast cancer. 2. Test if Six1-induced metastasis involves the TRAIL resistance mechanism, and 3. Determine if Six1 expression predicts TRAIL sensitivity and prognosis in patient tumors. Together, these aims should allow us to understand how Six1 regulates TRAIL receptor signaling, determine the role of these mechanisms in tumor metastasis and test if the same mechanisms apply in breast and ovarian cancer patients and thus determines their clinical outcomes. PUBLIC HEALTH RELEVANCE: Breast and ovarian cancer are the second and fifth leading causes of cancer death in women. One exciting new approach to treating these (and other) cancers is to use therapies that directly activate TRAIL receptors, however many tumor cells are resistant to these therapies. This project focuses on one mechanism (Six1 expression) by which tumor cells become resistant to TRAIL that our data indicate affect a high proportion of metastatic cancer patients (~60-90%). By understanding how this mechanism causes resistance to TRAIL, how this affects disease progression and metastasis and how this alters clinical outcomes in patients, we should gain new insights into the value of Six1 as a prognostic marker and better understand how to use the TRAIL therapeutics that are being developed.